Eukaryote-To-Eukaryote Gene Transfer Events Revealed by the Genome Sequence of the Wine Yeast Saccharomyces Cerevisiae EC1118

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Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118

Maite Novoa,1, Fre´de´ ric Bigeya,1, Emmanuelle Beynea, Virginie Galeotea, Fre´de´ rick Gavoryb, Sandrine Malletc, Brigitte Cambona, Jean-Luc Legrasd, Patrick Winckerb, Serge Casaregolac, and Sylvie Dequina,2

aInstitut National de la Recherche Agronomique, Unite´Mixte de Recherche 1083 Sciences Pour l’Oenologie, F-34060 Montpellier, France; bCommissariat a` l’Energie Atomique, Genoscope, F-91057 Evry, France; cInstitut National de la Recherche Agronomique, Unite´Mixte de Recherche 1238 Microbiologie et Geńe´ tique Molećulaire, Centre National de la Recherche Scientifique, AgroParisTech, F-78850 Thiverval-Grignon, France; and dInstitut National de la Recherche Agronomique, Unite´Mixte de Recherche 1131 Sante´de la Vigne et Qualite´du Vin, F-68021 Colmar, France

Edited by Jasper Rine, University of California, Berkeley, CA, and approved August 3, 2009 (received for review May 5, 2009) Saccharomyces cerevisiae has been used for millennia in winemak- variation between yeast strains. Comparative genome hybridiza- ing, but little is known about the selective forces acting on the wine tion analysis of several S. cerevisiae genomes has resulted in the yeast genome. We sequenced the complete genome of the diploid identification of gene deletions and amplifications common to commercial wine yeast EC1118, resulting in an assembly of 31 most wine yeast strains (7, 12). Analyses of the genome se- scaffolds covering 97% of the S288c reference genome. The wine quences of yeast strains of various origins have shown that yeast differed strikingly from the other S. cerevisiae isolates in nucleotide polymorphism may be the main source of phenotypic possessing 3 unique large regions, 2 of which were subtelomeric, variation (5, 6, 13Ϫ15). the other being inserted within an EC1118 chromosome. These With a view to deciphering the genetic basis of winemaking regions encompass 34 genes involved in key wine fermentation traits, we determined the complete genome sequence of the functions. Phylogeny and synteny analyses showed that 1 of these diploid commercial wine yeast strain EC1118. The full gene

regions originated from a species closely related to the Saccharo- repertoire of EC1118 was established and provided evidence GENETICS myces genus, whereas the 2 other regions were of non-Saccharo- for several gene transfer events, which were analyzed in detail. myces origin. We identified Zygosaccharomyces bailii, a major These findings provide unprecedented insight into the mole- contaminant of wine fermentations, as the donor species for 1 of cular mechanisms contributing to the adaptation of yeast to these 2 regions. Although natural hybridization between Saccha- winemaking. romyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non- Results Saccharomyces species. We show that the regions identified are Genome Sequence and Analysis. The diploid EC1118 genome was frequent and differentially distributed among S. cerevisiae clades, sequenced and assembled using a Sanger/pyrosequencing hybrid being found almost exclusively in wine strains, suggesting acqui- approach [supporting information (SI) Table S1 and SI Materials sition through recent transfer events. Overall, these data show and Methods]. Early in the assembly process it became clear that that the wine yeast genome is subject to constant remodeling distinct haplotypes could not be obtained in most cases, because through the contribution of exogenous genes. Our results suggest heterozygosity levels were very low (approximately 0.2%). An that these processes are favored by ecologic proximity and are 11.7-Mb high-quality ‘‘pseudohaploid’’ assembly with 31 scaf- involved in the molecular adaptation of wine yeasts to conditions folds was obtained (Table S1), corresponding to 96.7% of the of high sugar, low nitrogen, and high ethanol concentrations. S288c nuclear genome, as determined from genome alignments. Nucleotide alignments with other S. cerevisiae strains adaptive evolution ͉ comparative genomics ͉ horizontal gene transfer ͉ (Table S2) revealed a similar level of nucleotide polymorphism introgression ͉ Zygosaccharomyces bailii between EC1118 and S288c or the clinical isolate derivative YJM789 (46,825 and 47,253, respectively) and, as expected, a he yeast Saccharomyces cerevisiae has been associated with much lower level of nucleotide variation compared with the Thuman activity for thousands of years. The earliest evidence wine yeast derivatives RM11–1a and AWRI1631 (19,142 and of winemaking has been dated to Ϸ7,000 years ago (1). The 18,315, respectively). fermentation of grape juice exposes yeast cells to harsh envi- A total of 5,728 ORFs (except dubious and Ty-associated ronmental conditions (high sugar concentration, increasing al- genes) has been predicted for the nuclear genome of EC1118 cohol concentration, acidity, presence of sulfites, anaerobiosis, (Table S3), of which 5,685 are common to EC1118 and S288c. and progressive depletion of essential nutrients, such as nitrogen, Several of these ORFs are predicted to be affected by frameshifts vitamins, and lipids). These conditions, as well as unwitting selection by man for optimal winemaking traits (fermentation Author contributions: F.B., P.W., S.C., and S.D. designed research; M.N., F.B., E.B., V.G., F.G., performance, alcohol tolerance, and good flavor production) S.M., B.C., J.-L.L., and S.C. performed research; M.N., F.B., E.B., V.G., J.-L.L., S.C., and S.D. have generated hundred of strains that are currently used in the analyzed data; and M.N., F.B., S.C., and S.D. wrote the paper. wine industry. As a result, wine yeast isolates belong to a The authors declare no conflict of interest. well-defined lineage (2–6). This article is a PNAS Direct Submission. Deciphering the mechanisms that participate to these evolu- Freely available online through the PNAS open access option. tionary processes and identifying the variations contributing to Data deposition: The sequence reported in this paper has been deposited in the European the properties of wine yeast remain challenging issues. Wine Molecular Biology Laboratory database (accession nos. FN393058–FN393060, FN393062– yeasts are often diploid, heterozygous, and homothallic (4, 7, 8). FN393087, FN394216, and FN394217). They have a large capacity for genome reorganization through 1M.N. and F.B. contributed equally to this work. chromosome rearrangements (9–11), promoting rapid adapta- 2To whom correspondence should be addressed. E-mail: [email protected]. tion to environmental changes. Comparative genomics is a This article contains supporting information online at www.pnas.org/cgi/content/full/ suitable approach for cataloguing multiple types of sequence 0904673106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904673106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 (303 ORFs), in-frame stop codons (25 ORFs), or the absence of start or stop codons due to the presence of SNPs or indels (15 ORFs). We identified 11 Ty elements in the assembly (2 Ty1, 7 Ty2, 1 Ty4, and 1 Ty5), whereas 50 such elements have been identified in the S288c genome. We detected no Ty3 elements. This depletion of Ty elements is consistent with the results of comparative genome hybridization for the EC1118 strain (12). This overall picture was further supported by a direct estimate of the overall Ty abundance from sequencing reads (1.8%), much lower than that in S288c (3.4%), which was found to have the highest Ty abundance in a previous population study (5). This analysis also confirmed a clear inversion in the proportions of Ty1 and Ty2 in EC1118 compared with S288c. Fig. 1. Chromosomal distribution of the 3 unique EC1118 regions. The alignment of EC1118 contigs with S288c chromosomes led to the identiﬁca- Genes Present in S288c but Missing from EC1118. In total, 111 of the tion of 3 genomic regions unique to EC1118. The localization and length of genes present in S288c were not found in the EC1118 genome these 3 regions are indicated by colored chromosomal segments. The insertion (Table S4). Most of these genes are repeated and located in into chromosome VI of a 12-kb fragment from chromosome VIII is also shown. subtelomeric regions, which have not been accurately assembled, making it difficult to estimate copy number precisely. However, several of these genes (e.g., HXT16, PAU21, and SOR1) are known absent. An internal part of this region encompassing the genes to vary in copy number between strains (7, 12, 14). A large 17-kb from YFL059W to YFL062W (5 kb) was found inserted into the telomeric region on chromosome VI encompassing YFL052W to right telomeric end of chromosome X. Second, a 12-kb fragment YFL058W was absent in EC1118. Nontelomeric genes (21 genes) originating from chromosome VIII (including YHR211W to Ј were also found absent from EC1118. They consist mainly of genes YHR217C) was found in the 3 region of YFL051C (Fig. 1) that are present in tandem duplicated arrays (ENA2/5, MST27, resulting in YFL051C being fused to YHR211W (gene ࿝ ࿝ PRM8, ASP3, and FCY22) or in a 20.5-kb region of chromosome EC1118 1F14 0155g). The sequences of YFL051C and of XII adjacent to the rDNA array, including 4 copies of ASP3. Most YHR211W are highly similar, suggesting that the translocation of the missing nontelomeric genes were found frequently deleted in was mediated by homologous recombination. Similar transloca- other S. cerevisiae strains (Table S4). Two missing genes, MST27 and tions to chromosome X were also found in strains YJM789 and PRM8, belonging to the DUP240 family, have been found depleted RM11–1a. PCR, sequencing, and Southern blot analysis on in other wine yeasts (12, 16). EC1118 chromosomes confirmed these rearrangements. We identified a second unique region (region B) as a 17-kb Genes Present in EC1118 but Missing from S288c. We identified 34 insertion into chromosome XIV, between genes YNL037C and ORFs in EC1118, encoding proteins of 50 to 150 aa, that were YNL038W. Interestingly, a sequence similar to region B was absent from S288c. Only 6 of these ORFs were kept in EC1118 detected in the RM11–1a genome, but the sequence is slightly annotation (Table S3), thanks to the presence of identified rearranged compared with EC1118 and located between genes orthologs in S. cerevisiae strains YJM789, RM11–1a, and YNL248C and YNL249C. We confirmed the localization of AWRI1631 and conserved genomic sequences in Saccharomyces region B in EC1118 by PCR amplification of the breakpoints. sensu stricto: EC1118࿝1J19࿝0562g, present in most of these strains A third region, 65 kb in length (region C), was identified in the and species, EC1118࿝1G1࿝0023g, highly conserved in S. mikatae, subtelomeric region of the right arm of chromosome XV, the duplicated EC1118࿝1M36࿝0034g and EC1118࿝1M36࿝0045g, replacing the last 9.7 kb of this chromosome. Southern blot present in a single copy in S. mikatae and in AWRI1631 and analysis confirmed the location of region C on chromosome XV. in 2 copies in Schizosaccharomyces japonicus, and two other genes with a defined function. The first gene, KHR1 Function of the EC1118 ORFs Encompassed by the Unique Regions. (EC1118࿝1I12࿝1684g), which encodes a heat-resistant killer Within the three unique EC1118 regions, 34 ORFs predicted to toxin, is located in a 1.6-kb fragment inserted into EC1118 code for proteins of Ͼ150 aa in length and with homologs in chromosome IX and flanked by 2 LTR elements. KHR1 was also other species were identified (Table S5). These genes were found at the same location in the genome of YJM789. The classified according to the Munich Information Center for second gene, EC1118࿝1O30࿝0012g, is predicted to encode Mpr1, Protein Sequences (MIPS) functional catalog and were found to a protein with N-acetyltransferase activity conferring resistance be involved mostly in key functions of the winemaking process, to oxidative stress and ethanol tolerance (17). This ORF has such as carbon and nitrogen metabolism, cellular transport, and been identified in the ⌺1278b strain and, interestingly, also in the stress response (Fig. 2). other wine yeasts (RM11–1a and AWRI1631). During wine fermentation, yeast cells must convert large We also found another 34 genes and 5 pseudogenes to be amounts of glucose and fructose into alcohol. This process is also present in EC1118 but missing from S288c. Unlike the genes limited by nitrogen. Twenty of the 34 newly identified genes were described above, these genes were organized into 3 large clusters found to encode proteins potentially involved in the metabolism that have been analyzed in detail (see below). and transport of sugar or nitrogen. These genes included genes similar to those encoding a Kluyveromyces thermotolerans glucose Identification and Localization of Large Chromosomal Regions Unique transporter, the S. cerevisiae glucose high-affinity transporter to EC1118. Three large regions of the EC1118 genome, a total of HXT13, and the S. pastorianus–specific fructose symporter FSY1. 120 kb in length, which could not be aligned with the S288c Several of these genes have homologs with known functions in reference genome, were identified (Fig. 1). amino acid metabolism, such as a transcription factor involved in The first of these regions was 38 kb long (region A) and was proline utilization (PUT3), a S. cerevisiae permease potentially located in the subtelomeric region of the left arm of chromosome involved in the export of ammonia (ATO3), and 2 tandem- VI. The extremity of this chromosome displays a high degree of repeated genes encoding permeases of neutral amino acids. rearrangement (Fig. 1). A 23-kb fragment in the left arm of Another example of genes encoding proteins with nitrogen- chromosome VI (including YFL052W to YFL062W in S288c) is related functions is provided by the gene encoding 5-oxo-L-

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904673106 Novo et al. Downloaded by guest on September 26, 2021 Fig. 2. Functional classiﬁcation of the unique genes of EC1118. The potential functions of the 34 unique genes of EC1118 were deduced from their S. cerevisiae orthologs. EC1118 genes were clustered according to the MIPS functional catalog. Each category is represented in the chart by a color and a description of function. GENETICS

prolinase, which catalyzes the ATP-dependent cleavage of 5-oxoproline to give L-glutamate. We also identified 5 pseudogenes in subtelomeric regions A and C. In region A, we found a highly degenerate relic displaying sequence similarity to S. cerevisiae BIO3 and an intriguing pseudogene, EC1118࿝1F14࿝0067g, very similar to the AGL264W gene of Eremothecium gossypi encoding a bacterial transposase. These 2 genes do not encode hAT-like transposases, whereas gene 10980010 of AWRI1631 (15) and various Kluyveromyces genes encode proteins from this family (18). Three pseudogenes were identified in region C and shown to display similarity to S. Fig. 3. Analysis of the phylogeny and synteny of the genes in EC1118 region cerevisiae ARB1, SOR2, and NFT1. Rapid changes in coding B and in various yeast species. (A) Phylogram of the EC1118࿝1N26࿝0023g sequences leading to gene inactivation are more frequent at the homologs. Primary protein sequence alignment of EC1118࿝1N26࿝0023g and its telomeres in Saccharomyces (19), resulting in relics being largely homologs, searched using blastp in the National Center for Biotechnology concentrated in the subtelomeric regions (20), as observed here. Information and Geńolevures databases, was performed with ClustalX (47). Alignments were manually curated with GeneDoc (48). The unrooted boot- strapped neighbor-joining tree was built with ClustalX and visualized with Origin of the Unique Genes of EC1118. The existence of genes Treeview (49). Bootstrap values (percentages) based on 1,000 replicates are unique to EC1118 suggests the loss of these genes from other S. indicated at the nodes. A very similar tree was obtained using Phyml. (Scale cerevisiae strains or their acquisition from non–S. cerevisiae bar, 0.1 substitutions per site.) (B) The genomic localization and orientation of donors. Blastp analysis supported the second of these hypothe- orthologs of region B–specific genes are represented for the following species: ses, because the closest relatives were found in species belonging Z. rouxii (ZYRO, pale yellow arrows), Lachancea kluyveri (SAKL, gold arrows), to a clade containing the Lachancea, Zygosaccharomyces, K. thermotolerans (KLTH, pink arrows), Candida guilliermondii (CAGU, tur- quoise arrows), and Pichia sorbitophila (PISO, blue arrows). S288c orthologs Kluyveromyces, Saccharomyces, and Eremothecium genera (21) for the EC1118 genes flanking the region B are shown in light blue. Arrows (clade I) and species belonging to a large, recently reassessed represent ORFs and their orientation. Genes are identified by their name, and clade (22) containing Debaryomyces, some Pichia, and a number the chromosome or the scaffold to which they belong is shown by a letter of medically important Candida species (clade II) (Table S5). or a number within the arrow. In EC1118, N refers to the scaffold N26, and For accurate identification of the hypothetical donor species for in P. sorbitophila the numbers refer to the gene coordinates on the these genes, we carried out a combined phylogeny and synteny chromosome. Genes syntenic with those of EC1118 are shown as fully analysis. From these analyses, we observed different situations colored arrows. Gene order was analyzed with a genome browser for all for each region. species except P. sorbitophila, for which tblastn was used, because this genome is not yet annotated. Region A shows 2 different syntenic blocks: the first block has genes most closely related to Zygosaccharomyces rouxii genes, and the second block, whose synteny is conserved in species from both clade I and clade II, carries genes with their closest relatives observation, region B gene organization was rather well con- belonging to clade II species (Fig. S1). served with the related Zygosaccharomyces and Kluyveromyces Genes of region B were systematically grouped with Z. rouxii species (Fig. 3). The exception is EC1118࿝1N26࿝0034g, which in phylogenetic analysis, consistent with Z. rouxii genes being the only shows a good match to RM11–1a strain. best hits in blastp analysis (Fig. 3). In agreement with this Finally, genes in region C displayed some synteny with the genes

Novo et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 26, 2021 of species closely related to the Saccharomyces clade, consistent with the observed phylogenetic relationships (Fig. S2).

Donor Species of the Unique Regions. All natural hybrids discovered to date in Saccharomyces have involved species from the same genus (23–26). The phylogenetic analysis described above identified at least 1 potential donor of genetic material not closely related to Saccharomyces. We tried to identify the origin of the foreign genes found in EC1118, by carrying out PCR amplification with primers based on the sequences of genes from regions A, B, and C on genomic DNA isolated from strains (mostly type strains) belonging to 77 species from clade I or clade II (Table S6). Only the type strain of Zygosaccharomyces bailii CBS 680T gave positive results with primers based on region B. All of the primer pairs amplified specific fragments of the expected size. We therefore checked by PCR whether the organization of the various foreign genes detected in EC1118 was similar to that in Z. bailii. The organization of genes in region B was found similar in EC1118 and Z. bailii CBS 680T, with the exception of gene EC1118࿝1N26࿝0056g, which was located upstream from EC1118࿝1N26࿝0012g in Z. bailii, in an arrangement similar to that found in Z. rouxii (Fig. 3). The gene organization in Z. bailii was confirmed by sequencing a 14-kb nucleotide sequence (European Molecular Biology Laboratory accession no. FN295481) that was found to be 99.7% identical to that of EC1118, confirming the identification of Z. bailii as a donor of unique EC1118 genes. In addition, we also detected the presence of this region in 7 other strains of Z. bailii of various origins (Table S6). Analyses of phylogeny and synteny suggested that regions A and B might have a common origin (Figs. 3 and S1). However, no amplification was obtained when primers based on region A genes were used with DNA from Z. bailii. Similarly, no positive results were obtained for any of the other 46 species tested. The contributor of region A must therefore be an unidentified species related to clade I or II, as suggested by the 2 synteny blocks detected (Fig. S1). The presence, in region A, of a gene encoding a protein resembling a bacterial DNA transposase found only in the clade I species Eremothecium gossypii and the higher level of synteny with clade I than with clade II species strongly suggest that the contributor of region A belongs to clade I. No amplification was observed with primers based on the 16 genes of region C, with any of the 44 species tested (Table S6), including 26 species either found in the wine microflora or belonging to the group previously known as Saccharomyces sensu lato (21). The contributor of this region may be a non-described species very closely related to the Saccharomyces genus, as suggested by synteny and phylogeny analysis. Consistent with this hypothesis, we have shown that EC1118࿝1O4࿝6645g and EC1118࿝1O4࿝6656g (right end of region C), also found in strain AWRI1631, display some similarity to S288c telomeric Y’ ele- ment-encoded DNA helicases (Table S5). Y’ elements are only found in S. cerevisiae and in its closest relative, S. paradoxus (27). Thus, EC1118 contains gene clusters from Z. bailii and from 2 other species that we have not identified or that have not yet

at 11 loci, and is rooted according to the midpoint method. Source or geo- graphic origin of the strains is denoted by colored branches: green for wine, pink for sake, gold for oak and opuntia isolates (America), blue for fermented fruits (Malaysia and Netherlands), brown for palm and bili wine (Africa), violet Fig. 4. Dendrogram showing the presence of newly characterized genes for rum (French Indies), yellow for bread, pale brown for soil, pearl gray for among a set of 120 strains isolated from different sources. The 120 strains shown clinical, and orange for laboratory. The distribution of unique EC1118 regions here include the 35 strains of the S. cerevisiae Genome Resequencing Project (5). is represented by colored squares: yellow for region A, green for region B, pink The full name of each strain is available in Table S6. The neighbor-joining tree was for region C, and blue for the KHR1 gene. Half-ﬁlled squares indicate that at constructed from the Dc chord distance between strains based on polymorphism least 1 gene of the corresponding region is absent.

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904673106 Novo et al. Downloaded by guest on September 26, 2021 been described, one belonging to clade I and the other to the cific hybridization, and introgression (28). Recent analyses have Saccharomyces genus. shown that yeast hybrids may be more abundant in both natural and industrial environments than previously thought. Indeed, Distribution of the Unique Genes and Regions Among S. cerevisiae almost 10% of Saccharomyces strains previously classified as Strains of Different Origins. In a previous study of yeast diversity sensu stricto seem to be hybrids between different species (29). based on multilocus microsatellite typing, we showed that wine Lager brewing yeasts are natural hybrids generated by interspe- yeast strains clustered in a distinct phylogenetic group (4). We cific hybridization between S. cerevisiae, S. bayanus, and an as-yet investigated the distribution of unique EC1118 regions among non-described species (30–32). Double and triple hybrids of S. yeast populations, and particularly among wine yeasts, by car- cerevisiae with S. uvarum, S. kudriavzevii, or both were recently rying out PCR analysis on strains representative of the estab- identified in yeast populations isolated from grape and cider lished clades (4). fermentations [for a review see Sipiczki (33)]. However, most of A phylogenic tree based on 120 strains was obtained (Fig. 4), the hybrids described to date have either the genome of each including 66 wine strains and 19 isolates from various other parental species or chimeric genomes, and all of the donor origins (e.g., laboratory, palm wine, bakery, distillery, clinic, and species belong to the Saccharomyces genus. sake). The 35 strains recently sequenced by Liti et al. (5) were Horizontal gene transfers have rarely been described in yeast, also included in the analysis. and all previous examples have involved bacterial single genes Unique genes were searched in 53 strains, with a set of probes (13, 34, 35). The introgression of 23 kb from S. cerevisiae into S. used for each unique region. Region A was found in only 2 paradoxus has also been described (24) and resembled our groups—the Champagne group, containing EC1118-related findings, in particular for region C. The most likely explanation strains, and a closely related group containing flor yeasts— for this region is that a cross has occurred between S. cerevisiae suggesting that this region was acquired recently. Region B was and a Saccharomyces species to yield the present hybrid. It is found in the same groups and in more distantly related strains. generally thought that such hybrids are resolved by the gradual Region C was found to be as widespread as region B among S. loss of one of the contributing genomes (33). cerevisiae strains. Regions B and C were found to be incomplete The situation is clearly different for regions A and B, which in several strains, the missing genes differing between strains represent the first example of gene transfer between S. cerevisiae (Fig. 4). These data suggest that these regions are unstable in S. and non-Saccharomyces species. The phylogenetic topologies for

cerevisiae. genes of regions A and B indicate that the non-Saccharomyces GENETICS Most strains carrying the unique regions were closely related species are the donors and S. cerevisiae the recipient. The to the wine yeast group. Overall, regions B and C were found to presence of region B in Z. bailii strains from various origins be present in almost half the 53 strains tested. The 3 regions are further supports this hypothesis. Z. bailii is a major yeast exclusively (region A) or mostly (regions B and C) found in wine contaminant of wine. It tolerates common food preservatives, strains (29 of 35 wine strains contain at least 1 of the 3 unique high concentrations of sugar and ethanol, and low pH. These regions). The differential presence of the genes from regions A, properties confer on this species an outstanding capacity to B, and C in a number of wine yeast isolates may be accounted survive during wine fermentations. With S. cerevisiae,itisoneof for the progressive diffusion of these events by outcrossing inside the rare yeasts able to persist until the end of the fermentation the wine yeast population. The transfers of regions B and C seem process (36). It is therefore found in close contact with S. to be older than that of region A, but the timing of these events cerevisiae in many natural fermentations. This proximity may cannot be determined, because the subtelomeric location of have favored genetic transfer, either in a direct lateral transfer regions A and C is a source of instability. The KHR1 gene was or through introgression after hybridization. Although Z. bailii found to be widespread among strains, suggesting an ancient has been described as a diploid that does not undergo meiosis but acquisition event that has subsequently been lost from many produces tetrads with mitotic spores (37) neither of the 2 strains. The presence of LTR flanking KHR1 might account for hypotheses can be excluded. this instability. This strategy of evolution by gene transfer is an important To obtain a broader view of the distribution of regions A, B, aspect of yeast diversification and may play a major role in and C within the S. cerevisiae species, we performed a blastn adaptation to the wine fermentation ecosystem. Two of the survey of the unique genes in the genome of YJM789 (13), RM11–1a, AWRI1631 (15), and the 36 S. cerevisiae strains unique regions are located in subtelomeric regions, which are sequenced by Liti et al. (5) (Fig. S3). The region A was absent known to be enriched in genes involved in adaptation (29). In from all strains, consistent with the local distribution of this some situations, hybrids between species show increased fitness region in the ‘‘Champagne/Flor yeasts’’ cluster (Fig. 4). Region and acquire unique properties compared with the parental B was found in the wine yeast derivative RM11–1a, in strains of species. For example, hybrids between different Saccharomyces the clusters called ‘‘Wine/European’’ and ‘‘mosaic genomes’’ (5). species have been shown to grow over a broader range of Region C was also found in the latter strains, in only 1 strain temperatures or to produce larger amounts of glycerol or aroma belonging to the ‘‘Wine/European’’ cluster, and in strain compounds than the parental strains (25, 38, 39). The potential AWRI1631. The fact that this region was largely found in wine functions associated with the transferred genes, such as those yeasts in our PCR survey (Fig. 4) suggests that the ‘‘Wine/ related to fructose utilization, oxidative stress, or nitrogen European’’ group of Liti et al. (5) is not fully representative of metabolism, may contribute to the adaptation to the fermenta- the wine yeast community. It is also possible that because of tion of high-sugar, low-nitrogen grape musts and may confer a heterozygosity, some regions are present in the parental strains selective advantage during wine fermentation. but not in the derivative strains whose genome was sequenced. Materials and Methods The different distribution of regions B and C suggests that these regions have a different history. Interestingly, region C was Strains and Media. Lalvin EC1118 (EC1118), also known as ‘‘Prise de mousse,’’ almost exclusively found in strains of European origin. is a S. cerevisiae wine strain isolated in Champagne (France) and manufactured by Lallemand Inc. EC1118 has been deposited in the Collection Nationale de Discussion Cultures de Microorganismes (Institut Pasteur, France) as strain I-4215. This strain is one of the most frequently used fermentation starters worldwide and Various mechanisms are known to be involved in the adaptive has been extensively studied as a model wine yeast (40–42). The other yeast evolution of yeasts to the fermentation process, such as gene isolates used are detailed in Table S6. The non-Saccharomyces isolates were duplication, polyploidy, chromosomal rearrangements, interspe- obtained from the Centre International de Ressources Microbiennes-Levures

Novo et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 26, 2021 in France, and from the Centraalbureau voor Schimmelcultures in the Neth- Additional Materials and Methods. Further details are available in SI Materials erlands. Cells were routinely grown in YPD medium (1% yeast extract, 1% and Methods. peptone, and 1% glucose) at 28 °C, with shaking. ACKNOWLEDGMENTS. We thank Lallemand Inc. for providing the strain Gene Prediction and Annotation. Genome annotation was based on a combina- EC1118, Noemie Jacques for providing strains from the Centre Interna- tion of methods including ORF calling (minimum size, 150 bp), gene prediction tional de Ressources Microbiennes–Levures, and Giani Liti and Justin Fay for with GlimmerHMM (43), and direct mapping of S288c ORFs from the Saccharo- providing S. cerevisiae strains; the Geńolevures Consortium for providing myces Genome Database. The detailed annotation procedure and a complete free access to the genome of Pichia sorbitophila; Claude Gaillardin for annotation file are available in SI Materials and Methods and Table S3. helpful discussions and for providing Lebanese strains; Philippe Abbal for bioinformatics assistance; and Jean-Jacques Gonod for drawing figures. This work was supported by grants from the Consortium National de Microsatellite Analysis. The 120 strains were characterized for allelic variation Recherche en Geńomique-Genoscope (no. 2005/74 to S.D.) and the Bureau at 11 microsatellite loci, as described by Legras et al. (4). The chord distance Dc des Ressources Geńe´ tiques (no. 347 ‘‘Exploration de la diversite´fongique’’ between strains was calculated, as described by Cavalli-Sforza and Edwards to S.C.). M.N. and E.B. hold postdoctoral fellowships from the Generalitat (44). The neighbor-joining tree was constructed with the PHYLIP 3.67 package de Catalunya and Institut National de la Recherche Agronomique (INRA), (45) and drawn with MEGA software version 4.0 (46). The tree was rooted by respectively. This work was supported by INRA and Centre National de la the midpoint method. Recherche Scientifique.

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